of September 5, 2019.This information is current as

MutationZap70Systemic Autoimmunity Caused by

Strain-Specific Manifestation of Lupus-like

Ohmura, Takao Fujii and Tsuneyo MimoriMasao Tanaka, Hiromu Ito, Hajime Yoshifuji, Koichiro Tsuruyama, Kaoru Sakai, Hideki Yokoi, Mirei Shirakashi,Noriko Sakaguchi, Yoshinaga Ito, Masaki Hikida, Tatsuaki Takashi Matsuo, Motomu Hashimoto, Shimon Sakaguchi,

http://www.jimmunol.org/content/202/11/3161doi: 10.4049/jimmunol.1801159April 2019;

2019; 202:3161-3172; Prepublished online 24J Immunol 

MaterialSupplementary

9.DCSupplementalhttp://www.jimmunol.org/content/suppl/2019/04/23/jimmunol.180115

Referenceshttp://www.jimmunol.org/content/202/11/3161.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2019 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Strain-Specific Manifestation of Lupus-like SystemicAutoimmunity Caused by Zap70 Mutation

Takashi Matsuo,* Motomu Hashimoto,† Shimon Sakaguchi,‡ Noriko Sakaguchi,‡

Yoshinaga Ito,x Masaki Hikida,{ Tatsuaki Tsuruyama,‖ Kaoru Sakai,# Hideki Yokoi,#

Mirei Shirakashi,* Masao Tanaka,† Hiromu Ito,†,** Hajime Yoshifuji,* Koichiro Ohmura,*

Takao Fujii,†† and Tsuneyo Mimori*

A defect in TCR-proximal signaling is a major characteristic of CD4 T cells in systemic lupus erythematosus; however, it is not fully

known how defects in TCR signaling lead to lupus-like systemic autoimmunity characterized by germinal center development and

autoantibody production against nuclear Ags. In this study, we show that SKG mice, which develop autoimmune arthritis in a

BALB/c background due to defective TCR signaling by a Zap70 mutation, develop lupus-like systemic autoimmune disease in

the C57BL/6 (B6) background (B6SKG mice). B6SKG mice showed multiorgan inflammation with immune complex deposition

and anti-dsDNA Ab production. Follicular helper T cells (Tfh), which help germinal center formation, were spontaneously

expanded in B6SKG mice. Th cells secreting IFN-g or IL-17 and regulatory T cells were also increased in B6SKG mice compared

with wild-type B6 mice, with the regulatory T cell subpopulation losing the expression of CD25. Among the factors related to Tfh

differentiation, the number of dendritic cells and the expression levels of the costimulatory molecules CD80, CD86, and ICOSL in

dendritic cells but not in B cells were specifically increased in wild-type B6 mice compared with BALB/c mice. The inhibition of

these costimulatory molecules suppressed Tfh development and lupus-like autoimmunity. Thus, a defect in TCR-proximal sig-

naling leads to lupus-like systemic autoimmunity under the specific genetic background that facilitates Tfh development. The

Journal of Immunology, 2019, 202: 3161–3172.

Systemic lupus erythematosus (SLE) is a systemic auto-immune disease characterized by the production of auto-antibodies against nuclear Ags. Although the etiology of

SLE is multifactorial, CD4 T cells have been recognized to play afundamental role in its pathogenesis, contributing to the autoantibody

production of B cells (1). A defect in TCR signaling is a majorcharacteristic of SLE CD4 T cells (2). Multiple discrete signal-ing abnormalities in TCR signaling at the level of the TCRcomplex, cytosol, and nucleus have been reported in SLE CD4T cells (2). Among these, defects of TCR-proximal signalinginvolving the CD3-TCR complex and its associated ITAMs arethe most common abnormalities observed in SLE (3). In fact,the downregulation of the CD3z chain due to transcriptional orposttranscriptional defects has been detected in more than halfof SLE patients (4). Importantly, decreased expression levels ofthe TCRz chain in SLE persist regardless of disease activity ortreatments, in contrast with the transient reduction due to inflam-matory conditions observed in cancer, infection, or rheumatoidarthritis (RA) (4, 5). Similar to human studies, abnormalities ofvarious molecules in TCR signaling, such as CD45, Fyn, andZAP70, have been shown to result in the development of systemicautoimmune diseases in mice (6–9). However, the mechanism bywhich these defects in TCR signaling lead to systemic autoim-munity has not been fully addressed. Altered T cell selection inthe thymus (both positive and negative selection) and skeweddifferentiation of effector T cells and regulatory T cells (Tregs) inthe periphery have been indicated as potential mechanisms (8, 10).The SKG mouse strain with a BALB/c background (SKG) has

been used as a murine model of RA, which develops autoimmunearthritis under a nonspecific pathogen-free condition (8). Thecausative factor was identified as a point mutation (W163C) of theZap70 gene, located in the SH2 domain of ZAP70, which alters itsassociation with the ITAMs of the CD3z chain, thus reducing,but not abolishing, TCR signal transduction. The decrease in TCRsignaling due to the Zap70 mutation alters the thymic selectionthresholds so that T cells that strongly react with self-antigensfail to be deleted during thymic negative selection and escapeto the periphery. These self-reactive T cells then differentiate into

*Department of Rheumatology and Clinical Immunology, Graduate School of Medicine,Kyoto University, Kyoto 606-8507, Japan; †Department of Advanced Medicine forRheumatic Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8507,Japan; ‡Department of Experimental Immunology, Immunology Frontier ResearchCenter, Osaka University, Osaka 565-0871, Japan; xDepartment of Cancer Immunologyand Virology, Dana-Farber Cancer Institute, Boston, MA 02215; {Laboratory forMolecular Cell Physiology, Department of Life Science, Akita University, Akita010-8502, Japan; ‖Center for Anatomical, Pathological and Forensic Medical Research,Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; #Departmentof Nephrology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan;**Department of Orthopedic Surgery, Graduate School of Medicine, Kyoto University,Kyoto 606-8507, Japan; and ††Department of Rheumatology and Clinical Immunology,Wakayama Medical University, Wakayama 641-8510, Japan

ORCIDs: 0000-0003-3805-7224 (T. Matsuo); 0000-0002-3118-2826 (T.T.); 0000-0003-0803-0397 (K.S.).

Received for publication August 21, 2018. Accepted for publication March 27, 2019.

This work was supported by a Grant-in-Aid for Young Scientists (26860749) and aGrant-in-Aid for Scientific Research (16K09890) from the Ministry of Education,Culture, Sports, Science and Technology, Japan (to M. Hashimoto), and by a researchgrant from Bristol-Myers Squibb and ONO Pharma (to M. Hashimoto).

Address correspondence and reprint requests to Dr. Motomu Hashimoto, Departmentof Advanced Medicine for Rheumatic Diseases, Graduate School of Medicine, KyotoUniversity, 35 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mailaddress: mohashim@kuhp.kyoto-u.ac.jp

The online version of this article contains supplemental material.

Abbreviations used in this article: ANA, anti-nuclear Ab; DC, dendritic cell; DN,double-negative; DP, double-positive; GC, germinal center; Pdcd1, programmed celldeath protein 1; PTPN22, protein tyrosine phosphatase-22; RA, rheumatoid arthritis;SLE, systemic lupus erythematosus; SP, single-positive; Tfh, follicular helper T cell;Tfr, follicular Treg; Treg, regulatory T cell.

Copyright� 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

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IL-17–producing helper CD4 T cells (Th17) driven by homeostaticproliferation or by innate immune activation and result in thedevelopment of autoimmune arthritis in SKG mice (11, 12).Although SKG mice have a defect in TCR-proximal signaling,

and defects in TCR signaling are major abnormalities in SLE CD4T cells, SKG mice do not develop lupus-like systemic autoim-munity such as immune complex–mediated nephritis. SKG miceshow hypergammaglobulinemia and develop rheumatoid factor oran Ab against type-2 collagen but do not produce anti-dsDNA Ab(8). A potential explanation for this apparent contradiction may berelated to the specific genetic background of this mouse strainbecause BALB/c mice are resistant to the development of lupusin contrast to C57BL/6 (B6) mice in many autoimmune animalmodels. For example, deficiency of FcgRIIb or programmed celldeath protein 1 (Pdcd1) or the transgenic expression of B cellCLL/lymphoma 2 (Bcl2) all result in a lupus-like phenotype inthe B6 background but not in the BALB/c background (13–17).Although the specific mechanism that renders B6 animals moresusceptible to the lupus phenotype than BALB/c animals has notbeen fully defined, several mechanisms have been proposed, suchas a difference in receptor editing of the Ig L chain (18), skewingto Th1 cells (19), or increased type-1 IFN, which are all keymediators in the pathogenesis of SLE (20, 21).In this study, we demonstrate that SKG mice with a B6 back-

ground (B6SKG) develop lupus-like systemic autoimmunity withmarked expansion of follicular helper T cells (Tfh) and germinalcenter (GC) development.We propose that an increase in the numberof dendritic cells (DCs) with higher expression of costimulatorymolecules in the B6 strain compared with the BALB/c strain mayfacilitate the strain-specific development of Tfh and lupus in B6SKGmice. Thus, a defect in TCR-proximal signaling may predisposeindividuals to autoimmunity, whereas the specific genetic backgroundmay provide additional coactivating components necessary for thedevelopment of systemic autoimmune disease.

Materials and MethodsMice

BALB/c A (BALB/c), C57BL/6J (B6 wild-type [B6WT]), and SKG micewere purchased from CLEA Japan. SKG mice were backcrossed to B6WTmice for eight generations to generate C57BL/6ZAP70 skg/skg (B6SKG)mice. All mice were maintained in a specific pathogen-free condition inour animal facility in accordance with the guidelines for animal care ap-proved by Kyoto University. Age- and sex-matched mice were used for allexperiments.

Histology

The salivary gland, thyroid gland, thymus, lung, heart, stomach, ileum,intestine, liver, pancreas, kidney, spleen, skin, and ankle joint were fixed inbuffered 10% formalin, and paraffin-embedded sections were stained withH&E. Stained sections were visualized using a system microscope (BX43and DP26; Olympus) and were evaluated by a single pathologist (T.T.).

Immunohistochemistry

Frozen 5-mm sections of the kidneys from B6WT and B6SKG mice weresubjected to immunohistochemical staining. The kidney section wasstained with IgG (Jackson ImmunoResearch) and C3 (Cappel). The sec-tions were fixed with acetone (Wako) and stained with the indicatedAbs in PBS. Finally, the sections were washed and mounted with Vecta-shield (Vector Laboratories), and stained sections were visualized using anall-in-one fluorescence microscope (BZ-700; Keyence Co.). The Crithidialuciliae assay was conducted with the Fluoro nDNATest kit (MBL) usingmouse anti-IgG (Jackson ImmunoResearch).

Frozen 10-mm sections of the spleens from B6WT and B6SKG micewere stained with anti-PNA (Vector Laboratories), anti–GL-7 (GL-7;BD Biosciences), and anti-CD38 (90; eBioscience) Abs. The sectionswere fixed with acetone (Wako), blocked with Blocking One (NacalaiTesque), and stained with the indicated Abs in PBS. Finally, the sectionswere washed and mounted with SlowFade Gold (Invitrogen), and the stained

sections were visualized using an LSM 710 NLO confocal fluorescent mi-croscope and analyzed with ZEN software (Zeiss). The immunofluorescencepattern of the anti-nuclear Ab (ANA) was evaluated by the Hep-2 ANA test(MBL) using mouse anti-IgG (Jackson ImmunoResearch).

ELISA

ANA-IgM and IgG levels were evaluated by the MESACUP ANA test(MBL) using the anti-mouse HRP-IgM Ab (Southern Biotech) and anti-mouse HRP-IgG Ab (Promega). Anti-dsDNA Ab IgG in the serum wasevaluated by a mouse anti-dsDNAELISA kit (Shibayagi). Total IgG, IgG2b,IgG2c, IgG3, and IgM levels were evaluated by the mouse ELISA Ready-Set-Go kit (eBioscience). IgG1 levels were evaluated by a mouse IgG1ELISA kit (Bethyl Laboratories).

Flow cytometry

The following Abs were purchased from BioLegend: anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CXCR5 (L138D7), anti–PD-1(29F.1A12), anti-ICOS (C398.4A), anti-B220 (RA3-6B2), anti–GL-7(GL-7), anti–IL-17 (TC11-18H10), anti–IFN-g (XMG1.2), anti-CD44(IM7), anti-CD62L (MEL-14), anti–I-Ad (39-10-8), and anti–I-Ab(AF6-120.1). The following Abs were purchased from eBioscience: anti-CD25 (7D4), anti-Foxp3 (FJK-16s), and anti-AA4.1 (AA4.1). The followingAbs were purchased from BD Bioscience: anti-CD95 (Jo2) and anti–Bcl-6(K112-91). The transcription factors Foxp3 and Bcl-6 were stained afterfixation and permeabilization (eBioscience). For intracellular cytokine stainingof IL-17 and IFN-g, spleen cells were stimulated with 20 ng/ml PMA (Sigma-Aldrich) and 1 mM ionomycin (Sigma-Aldrich) in the presence of GolgiStop(BD Biosciences) for 4 h before staining. Costimulatory molecules on splenicDCs were evaluated 24 h after purification by MACS LS columns (MiltenyiBiotec) using CD11c (N418) beads (22). Flow cytometric acquisition wasperformed on a FACSCalibur system (BD) and analyzed with LSRFortessa(BD). Analysis was performed using FlowJo and WinMDI software.

Proliferation assay

CD4 T cells from the spleen were purified in MACS LS columns (MiltenyiBiotec) using CD4 (RM3-4) beads. Purified cells were loaded with theCFSE Cell Proliferation Kit (Invitrogen), washed, and incubated with plate-bound anti-CD3 mAb (10 mg/ml) and soluble anti-CD28 mAb (1 mg/ml)for 3 d. Cells were harvested and analyzed by flow cytometry.

Real-time PCR

CD4 T cells and CD11chigh DCs isolated from the spleen cells were purified byMACS LS columns (Miltenyi Biotec) using CD4 (RM3-4) and CD11c (N418)beads according to the manufacturer’s instructions. The purity was above 95%for total CD4 T cells and above 75% for total CD11chigh DCs. The purified cellswere lysed, and the RNAwas extracted using the RNeasy Micro Kit (Qiagen)and converted into cDNAwith the High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems) using Mastercycler (Eppendorf). Cytokine and tran-scription factor expression levels were assessed by real-time quantitative PCRusing a TaqMan gene expression assay probe and TaqMan Gene ExpressionMaster Mix run on the 7500 Real-Time PCR system (Applied Biosystems).Expression levels were normalized by the housekeeping gene Hprt.The following primers were purchased from Applied Biosystems: Il-6(Mm00446190_m1), Il-21 (Mm00517640_m1), andHprt (Mm01545399_m1).

Experimental treatments

For blockade experiments, 8–10-wk-old B6SKGmicewere i.p. injected with thespecific Abs indicated below, and spleen cells were obtained after 2 wk. Anti-OX40L (RM134L, 100 mg; BioLegend) and anti-ICOSL (HK5.3, 100 mg;BioLegend) were injected i.p. every 3 d (a total of five times for 2 wk) in a totalof 500 mg. CTLA4 Ig (Abatacept; Bristol-Myers Squibb) was injected once at500 mg. For evaluating immune complex deposition in the kidney after CTLA4-Ig treatment, 125 mg of CTLA4-Ig was injected once a week for 8 wk (total1000 mg). For the TLR stimulation experiment, the mice were s.c. implanted for14–28 d with an osmotic pump (no. 1002, 1004; DURECT) containing thefollowing TLR agonists: 700 mg of Poly(I:C) (P9582; Sigma-Aldrich), 280 mgof LPS (L4391; Sigma-Aldrich), 280 mg of imiquimod (sc-200385; Santa CruzBiotechnology), and 280 mg of CpG-ODN1668 (Hokkaido System Science).Zymosan A (Z4250; Sigma-Aldrich) and mannan (M7504; Sigma-Aldrich)were injected i.p. twice a week for 4 wk for up to 2 and 20 mg, respectively.

Assessment of arthritis score

The arthritis score was assessed as described previously (23). In brief,swelling of the finger joint received a score of 0.1, and swelling of the wristand finger joints received scores of 0.5–1.0.

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Statistics

We used a two-sided Student t test for all statistical analyses. A p value,0.05 was considered statistically significant.

Study approval

All experimental procedures were performed in accordance with the ethicalguidelines of Kyoto University. This study was approved by the AnimalResearch Committee, Graduate School of Medicine, Kyoto University(Medkyo 16106).

ResultsDevelopment of lupus-like systemic autoimmune disease inB6SKG mice

SKG mice were backcrossed for eight generations with B6WTmice, and their organs were examined histologically. The tissuesof B6SKG mice were normal or showed only mild abnormalityat 3 mo of age, whereas obvious abnormalities were found at 12 moof age. B6SKG mice showed normal histology of the kidney

FIGURE 1. Development of lupus-like systemic autoimmune disease in B6SKG mice. (A–C) H&E staining of tissue sections of B6WTand B6SKG mice

at 3 and 12 mo of age. (A) Kidney section. (B) Lung section. (C) Salivary gland section. (D) Major diameter of the glomeruli. Ten glomeruli at the middle

cortex area per mouse at original magnification 3400; (n = 3 each) **p , 0.01 versus control mice, Student t test. (E) Immunohistochemistry of glomeruli

at 3 mo of age for IgG and complement C3. Scale bars in (A)–(C) and (E), 20 mm.

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compared with B6WT mice at 3 mo of age, whereas destructionof vascular endothelial cells, a relative increase in the mesangiumarea, and atrophy of the glomeruli were observed in 12-mo-old

B6SKG mice compared with age-matched B6WT mice (Fig. 1A,1D). In the lungs, there was mild subbronchial accumulationof lymphocytes detected at 3 mo, which increased at perivascular,

FIGURE 2. Serology of B6SKG mice. (A) Immunofluorescent staining of ANAwith permeabilized Hep-2 epithelial cells at original magnification3400.

The serum samples were diluted 80 times. The nucleus of Hep-2 cells was stained with the sera of B6SKG mice and B6WT mice. Scale bar, 10 mm.

(B) ELISA for ANA-IgM and ANA-IgG. The ELISA plate was coated by a mixture of nuclear Ags, including Sm, RNP, histone, SS-A, and dsDNA. The

serum samples were diluted 100 times (n = 10 each). (C) ELISA for serum IgM (330,000 dilution) and IgG (3100,000 dilution). (D) ELISA for serum

IgG1, IgG2b (320,000), IgG2c (310,000), and IgG3 (330,000) levels (n = 10 each). (E) ELISA for anti-dsDNA Ab (351 dilution) (n = 20 each).

(F) Crithidia luciliae assay at original magnification 3200. *p , 0.05, **p , 0.01 versus control mice, Student t test.

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FIGURE 3. Spontaneous development of the GC in B6SKG mice. (A) H&E staining of the spleen. Clear distinction between red pulp and white pulp was

observed in B6WTmice but not in B6SKGmice at original magnification340. (B) Immunohistochemistry of the spleen with staining for PNA (blue), CD38 (green),

and GL-7 (red); scale bar, 100 mm. (C) Flow cytometric analysis of GL7 and FAS gated on B220+AA4.12mature B cells (n = 10 each). (D) Proportion of B220+ B

cells in splenic lymphocytes. (E) Proportion and number of GL7+ FAS+ GC B cells among B220+ AA4.12 B cells (n = 10 each). (Figure legend continues)

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stromal, and intraepithelial lesions, leading to the remodeling of thebronchus at 12 mo of age in B6SKG mice (Fig. 1B). In the salivaryglands, signs of sialadenitis (accumulation of lymphocytes, edematouschange, and destruction of the salivary ducts) were observed inB6SKG mice at 12 mo of age (Fig. 1C). B6SKG mice also showedabnormalities in the joints, pancreas, and skin (Supplemental Fig. 1),whereas other tissues, including the thyroid gland, heart, liver,stomach, and intestine, did not show major abnormalities inB6SKG mice compared with B6WT mice.Because B6SKG mice showed signs of inflammation in multiple

tissues, including the kidney, lung, and salivary glands, we furtherinvestigated whether these changes were mediated by immunecomplexes. Even in the 3-mo-old B6SKG mice that showedminimal abnormality in the glomeruli compared with B6WT mice,immunofluorescence analysis of the kidney revealed predominantdeposition of IgG in the glomeruli, unlike B6WT mice (Fig. 1E).Similarly, complement C3 deposition in the glomeruli was observedonly in B6SKG mice and not in B6WT mice. The deposition ofIgG and C3 in the renal glomeruli strongly suggested that theglomerulonephritis was immune complex mediated (Fig. 1E).Next, to determine whether the nephritis in B6SKG mice is

driven by lupus-like systemic autoimmunity, we assayed the serafor the presence of autoantibodies against nuclear Ags. Immu-nofluorescent staining of permeabilized Hep-2 epithelial cellsrevealed the presence of ANA, with a peripheral staining patternin the sera of 3-mo-old B6SKG mice, whereas no such reactivitywas detected in B6WT mice of the same age (Fig. 2A). ELISAfor ANA (coated with Sm, histone, SS-A, and other Ags) revealedan increased titer of ANA-IgG in B6SKG mice compared withB6WT mice, whereas the titer of ANA-IgM did not differ be-tween the two strains (Fig. 2B). Moreover, the total amountsof serum IgM and IgG were higher in B6SKG mice than inB6WT mice (Fig. 2C), suggesting the development of hyper-gammaglobulinemia. Among the IgG subsets, the titers of IgG2b,IgG2c, and IgG3, but not IgG1, were significantly elevated inB6SKG compared with B6WT mice (Fig. 2D), similar to theserological characteristics of Pdcd1 knockout mice (14). ELISArevealed that the titer of anti-dsDNA Ab IgG was markedly el-evated in B6SKG mice compared with that in BALB/c, SKG,and B6WT mice (Fig. 2E). The specificity for dsDNA was alsoconfirmed by a Crithidia luciliae assay (9). The sera of anti-dsDNA Ab-positive B6SKG mice reacted with the kinetochore(which is free of ssDNA and not complexed to a protein),thereby confirming the reactivity against dsDNA, whereas onlynonspecific binding was observed in the sera of B6WT, BALB/c,or SKG mice (Fig. 2F).Because systemic autoimmunity can be accelerated by innate

immune stimulation (24), we stimulated B6SKG mice with TLRagonists, which have been reported to trigger lupus in otheranimal models (25–28). We selected 2–3-mo-old B6SKG micefor this experiment, which showed a low titer of anti-dsDNAAb (,100 mU/ml) by ELISA as well as control B6WT mice,and both groups were chronically stimulated with various TLRagonists. The anti-dsDNA Ab level significantly increased aftertreatment with imiquimod (TLR7 agonist) or CpG (TLR9 ag-onist) and to a lesser extent after Poly(I:C) (TLR3 agonist) orLPS (TLR4 agonist) treatment in B6SKG mice (SupplementalFig. 2A). Treatment with zymosan or mannan, which triggers

autoimmune arthritis in SKG mice, only slightly elevated the levelof anti-dsDNA Ab, whereas mannan but not zymosan treatmenttriggered mild arthritis in B6SKG mice (12, 23) (SupplementalFig. 2B).Collectively, these results demonstrate that B6SKG mice de-

velop lupus-like systemic autoimmune disease with immunecomplex deposition and anti-dsDNA Ab production, which can beaccelerated by innate immune stimulation.

Spontaneous development of the GC and expansion of Tfh inB6SKG mice

Because the production of autoantibodies is highly associated withthe formation of the GC, we analyzed the development of theGC in the spleen of B6SKG mice. B6SKG mice showed mildsplenomegaly compared with B6WT mice (data not shown).H&E staining of the spleen further revealed effacement of thenormal architecture (clearly defined areas of red and whitepulp) in the spleen of B6SKG mice, suggesting a hyperreactivefeature (Fig. 3A). Immunohistochemistry of the spleen showedthe spontaneous development of PNA+ CD382 GL-7+ GC areas inB6SKG mice but not in B6WT mice (Fig. 3B).GC development in B6SKG mice was also confirmed by flow

cytometry. In B6SKG mice, the proportions of GL7+ FAS+ GCB cells were significantly increased among B220+ AA4.12 matureB cells, and the numbers of both GC B and FAS+ GL72 B cellswere also increased (Fig. 3C). The proportion of total B cells andboth the proportion and total number of GC B cells increased inB6SKG mice (Fig. 3D, 3E).Because GC formation is mediated by Tfh, we further examined

the proportion of Tfh, characterized by the expression of thechemokine receptor CXCR5 and the transcription factor BCL6as well as by higher expression levels of PD-1 and ICOS. Theproportion of CXCR5+ Bcl6+ Tfh was significantly increased inB6SKG mice compared with that in B6WT, BALB/c, or SKGmice (Fig. 3F). Although B6SKG mice had a reduced numberof CD4 T cells due to the alteration of thymic selection (Fig. 3G),similar to SKG mice, the proportion and total number of Tfh in-creased in B6SKG mice compared with those of B6WT mice(Fig. 3H). Splenic CD4 T cells in B6SKG mice expressed highlevels of PD-1 and ICOS (Fig. 3I), and Il-21 mRNA expressionwas highly elevated in the CD4 T cells of B6SKG mice (Fig. 3J),consistent with the increased development of Tfh.Because the Zap70 gene is primarily expressed in T cells, we

analyzed thymic T cell selection and effector T cell and Tregdevelopment more precisely in B6SKG mice (Fig. 4). The medullaof the thymus was atrophic in B6SKG mice, suggesting defects inthymocyte development (Fig. 4A). Because pre-TCR/TCR sig-naling via ZAP70 is involved in the passage from the double-negative (DN)3 (CD25+ CD442) to DN4 (CD252 CD442) stageand from the double-positive (DP) to single-positive (SP) stage,B6SKG mice showed a reduction in the frequency of DN4 cellswith a corresponding increase in DN3 cells as well as a reductionin CD4 SP and CD8 SP cells with a corresponding increase in DPcells compared with B6WT mice (Fig. 4B), similar to findings forSKG mice with other genetic backgrounds (29). In the periphery,B6SKG mice showed decreased populations of both CD4 andCD8 T cells (Fig. 4C). CD4 T cells from B6SKG mice werehypoproliferative against in vitro anti-CD3/CD28 stimulation

(F) Flow cytometric analysis of CXCR5 and Bcl6 gated on CD4 T cells. (G) Proportion of CD4 T cells in splenic lymphocytes (n = 10 each). (H) Proportion

and numbers of Tfh (n = 10 each). (I) Flow cytometric analysis of PD-1 and ICOS gated on CD4 T cells. (J) Quantitative real-time PCR for Il-21 mRNA of

splenic CD4 T cells (n = 3 per group). Flow cytometric data (C, F, and I) are representative of at least three independent experiments. **p , 0.01 versus

control mice, Student t test.

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FIGURE 4. Thymic selection: Th1, Th17, and Tregs in B6SKG mice. (A) H&E staining of the thymic gland in 28–35-d-old B6WT and B6SKG mice.

(B) Flow cytometric analysis of the maturation of developing T cells in the thymus (n = 5 each). The percentages of DN3 and DN4 were expressed as the

percentage of DN3 and DN4 relative to total DNs. The percentages of DNs, DPs, and SPs were assessed on the basis of the percentage of total thymocytes.

(C) CFSE dilution assay of CD4 T cells purified from B6WT and B6SKG mice. Proliferation of the CD4 T cells was assessed (Figure legend continues)

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due to the Zap70 mutation (Fig. 4D). However, the proportion ofCD62+ CD442 naive CD4 T cells decreased, whereas the pro-portions of CD44low CD62L2 effector and CD44high CD62L2

memory T cells increased in B6SKG mice compared with B6WTmice (Fig. 4E) because lymphopenia and homeostatic prolif-eration drives their differentiation into the effector/memoryphenotype without exogenous Ag exposure. These developmentaldefects in thymocytes, CD4 T cell lymphopenia, and spontaneousdifferentiation into effector/memory phenotype were comparable inSKG mice with the BALB/c background (Supplemental Fig. 3).Among the CD4 Th cell subsets, the proportion of Th17 cells washigher in B6SKG than that in B6WT mice (Fig. 4F, 4G), similar tofindings for SKG mice (11). The proportion of Th1 cells was alsohigher in B6SKG mice than that in B6WT mice, with no differencebetween SKG and BALB/c mice (Fig. 4F, 4G), consistent with theknown Th1 bias in the B6 strain (19). The proportion of Foxp3+

Tregs was higher in B6SKG mice than that in B6WT mice (Fig. 4F,4G), similar to the previously reported increase in Tregs in SKGmice compared with BALB/c mice (10). Interestingly, among theFoxp3-positive cells, the proportion of CD252 Foxp3+ CD4 T cellswas markedly increased in B6SKG mice (Fig. 4F).We also examined the proportion of CXCR5+ Bcl-6+ Foxp3+

follicular Tregs (Tfr), which suppress Tfh activity in the GC (30–32).The proportion of Tfr among CD4 T cells was increased in B6SKGmice, whereas the Tfr/Tfh ratio was not significantly differentbetween B6SKG and B6WT mice (Fig. 4H). Interestingly,B6SKG mice had increased more CD252 Tfr and fewer CD25+

Tfr compared with B6WT mice (Fig. 4I).Collectively, these findings show that B6SKG mice exhibit

abnormal thymic T cell development, CD4 T cell lymphopenia, andspontaneous differentiation into effector and memory CD4 T cells,including Th1 and Th17 cells, along with an increase in Tregs. Inaddition, the loss of CD25 in the subpopulations of Tregs and Tfr isa characteristic feature of B6SKG mice.

Factors facilitating the strain-specific development of Tfh

To gain insights into the molecular mechanisms that facilitated thestrain-specific expansion of the Tfh population in B6SKGmice, wecompared the total number, cytokine production, and costimulatorymolecule expression in DCs and B cells between wild-type BALB/cand B6 mice, which have been reported to regulate Tfh de-velopment (33). The proportion of CD11chigh DCs in the spleenwas significantly higher in B6WT mice than in BALB/c mice(Fig. 5A), whereas Il-6 mRNA expression in DCs was not sig-nificantly different between B6WT and BALB/c mice (Fig. 5B).Notably, the expression levels of the costimulatory moleculesCD80/86 and IOCSL but not OX40L were significantly higher inthe DCs of B6WT mice than in those of BALB/c mice (Fig. 5C,Supplemental Fig. 4), consistent with the reported upregulation ofCD86 in B6 DCs (22). In contrast, the expression levels of thesecostimulatory molecules in B cells were not different betweenBALB/c and B6WT mice (Fig. 5D). The number of DCs, Il-6mRNA expression levels in DCs, and costimulatory moleculeexpression levels in DCs and B cells were also compared be-tween mice with wild-type Zap70 and mutated Zap70 mice in aBALB/c and B6 background (Supplemental Fig. 4). The B6 mouse

strain had a higher number of DCs with higher expression levels ofcostimulatory molecules compared with the BALB/c strain.To test whether the expression of these costimulatory molecules

is involved in Tfh differentiation, we treated B6SKG mice withAbs blocking CD80/86 (CTLA4-Ig), ICOSL, and OX40L. Theproportions of Tfh and GC B cells were significantly decreasedafter CTLA4-Ig or anti-ICOSL treatment (Fig. 5E, 5F). This resultsuggested that interaction with these costimulatory moleculesplays an important role in the expansion of the Tfh population inB6SKG mice.Finally, we examined whether the inhibition of CD80/86 by

CTLA4-Ig could reduce Tfh and GC development and subse-quently inhibit immune complex deposition in the kidney in B6SKGmice. After repetitive treatment with CTLA4-Ig, IgG and C3deposition in the glomeruli of kidney sections was significantlyreduced, suggesting that these costimulatory molecules are indeedinvolved in Tfh expansion and the development of lupus-likenephritis in B6SKG mice (Fig. 5G, 5H).

DiscussionIn this study, we demonstrated that a defect in TCR-proximalsignaling caused by a Zap70 mutation specifically predisposesmice to the development of both arthritis and lupus, depending onthe genetic background. In the BALB/c background, the Zap70mutation causes a Th17-dependent form of autoimmune arthritis(11, 12), whereas in the B6 background, the same mutation causeslupus-like systemic autoimmune disease with an expansion of theTfh population. Although the mechanism that leads to the pref-erential development of lupus in the B6 background may bemultifactorial, the increased number of CD11chigh DCs withhigher expression of costimulatory molecules in the B6 strainmight be one factor that has facilitated the differentiation of Tfhand the development of lupus-like systemic autoimmune diseasein B6SKG mice. These results suggest that the pathogenic effectof a defect in TCR-proximal signaling depends on the complexinteraction with genetic and environmental factors, which maydetermine the ultimate manifestation of the disease.Although the disease in B6SKG mice is less severe than that

observed in the classical models such as NZB/W F1 or MRL/lprmice, it is more akin to the lupus-like diseases that occur in severalother genetically engineered mice, such as Pdcd1 knockout miceor Bcl2 transgenic mice, in which significant early mortality orovert proteinuria has not been reported (14, 16). One of the maincharacteristics of B6SKG mice is the spontaneous developmentof Tfh and the GC, which has also been reported in other lupusanimal models (34, 35). An increase in the number of Tfh-likecells in the peripheral blood has also been reported in humanSLE patients. Because defects in TCR-proximal signaling andTfh expansion are also common findings in human SLE patients(36), B6SKG is a suitable model to study how TCR signalingdefects contribute to the development of lupus.A critical question remaining is why lupus-like systemic auto-

immune disease developed only in B6SKG mice and not in SKGmice with the BALB/c background. The strain-specific develop-ment of lupus in the B6 strain but not in the BALB/c strain hasbeen observed in other lupus animal models (13–17). Thus, the B6

on day 3. (D) Flow cytometric analysis of CD4 and CD8 on lymphocytes in the spleen. (E) Flow cytometric analysis of CD44 and CD62L gated on CD3+

CD4+ T cells in the spleen. (F) Flow cytometric analysis of IL-17 and IFN-g gated on CD4 T cells (upper). Flow cytometric analysis of CD25 and Foxp3

gated on CD4 T cells (lower). (G) Proportion of Th1, Th17, and Tregs (n = 5 each). (H) Proportion of CXCR5 and Bcl6 coexpressing Foxp3+ cells (Tfr) in

the spleen. The proportion of Tfr (CXCR5+ Bcl6+ Foxp3+) among CD4 T cells and ratio of Tfr (CD4+ CXCR5+ Bcl6+ Foxp3+) to Tfh (CD4+ CXCR5+

Bcl6+) are shown (n = 5 each). (I) Proportion of CD25+ Tfr and CD252 Tfr (n = 5 each). Flow cytometric data (D, E, and F) are representative of at least

three independent experiments. **p , 0.01 versus control mice, Student t test.

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FIGURE 5. Factors facilitating the strain-specific expansion of Tfh. (A) Proportion of CD11chigh DCs in the spleen (n = 5 each). (B) Il-6 mRNA expression of

splenic CD11c DCs (n = 3 each). (C) Histograms of CD80, CD86, ICOSL, and OX40L expression in CD11chigh DCs. BALB/c (blue-filled histogram), B6WT (pink-

filled histogram), and isotype control (gray line). (D) Histograms of CD80, CD86, ICOSL, and OX40L expression in B cells. BALB/c (blue-filled histogram),

B6WT (pink-filled histogram), and isotype control (gray line). (E) Flow cytometric analysis of CXCR5+ Bcl6+ Tfh and GL7+ FAS+ GC (Figure legend continues)

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strain, which is normally considered to be disease-free and in-nocuous in genetic analyses, may nevertheless harbor certain locithat predispose mice to the development of systemic autoimmunedisease. The causes for these strain differences in the suscepti-bility to lupus have been studied, and some candidates have beenreported, including receptor editing of the Ig L chains (18), biasto a Th1 response (19), or increased type-1 IFN in B6 mice (20).One major reason for the difference in the disease manifestationbetween B6SKG and SKG mice might be the difference in MHCclass II molecules and repertoire selection because SKG micewith a C57BL/6 MHC H2-Aq (B6.Q) background develop ar-thritis but not lupus, although the production of anti-DNA Abs orimmune complex deposition in tissues was not examined (29). Inaddition to these factors, we suggest that the increased numberof CD11chigh DCs with higher expression of the costimulatorymolecules CD80, CD86, and ICOSL in the B6 strain might beone of the causes that facilitated the differentiation of Tfh andthe development of lupus-like systemic autoimmune disease inB6SKG mice.Differentiation of Tfh is a multistage, multifactorial process

characterized by initial priming by the interaction with DCs andfurther commitment by the interaction with B cells (33). Tfhdifferentiation is determined by the strength of TCR signaling,the specific cytokines milieu (including IL-6 and IL-21), and theexpression level of costimulatory molecules that interact withTCR signaling (33). Although IL-6 and IL-21 contribute to Tfhdifferentiation, their roles are redundant because both Il6 andIl21 knockout mice show normal Tfh or GC development (37).By contrast, both Cd28 and Icos knockout mice fail to developTfh or the GC (38, 39), suggesting the critical importance ofcostimulatory molecules for the development of Tfh. The inter-action of CD80/86 with DCs is required for the initial primingof Tfh as well as other helper CD4 T cell subsets (38, 40). TheICOS2ICOSL interaction at the time of DC priming is requiredfor the expression of Bcl6 on CD4 T cells, and the ICOS2ICOSLinteraction with B cells further upregulates Bcl6 expression (39,41, 42). Although Ox40 knockout mice show normal Tfh devel-opment, the OX40–OX40L interaction induces CXCR5 expressionand is involved in CD4 T cell migration to the T–B cell borderafter priming (43). Thus, costimulatory molecules are criticallyimportant for the development of Tfh, and multiple signals actingin concert, including CD80/86, ICOSL, and OX40L, at the time ofDC priming are required for the initiation of Tfh differentiation,consistent with previous reports (33).In SKG mice, a TCR signaling defect alters the thymic selection

thresholds, and T cells that strongly react with self-antigens are noteliminated in the thymus and thus escape into the periphery (8).However, the activation and proliferation of these self-reactiveT cells upon TCR stimulation is severely impaired because ofthe equal decrease in TCR signaling due to the Zap70 mutation.Unlike other Th cell subsets, Tfh differentiation requires thestrongest level of TCR signaling and continuous interaction withDCs (44). Therefore, T cells with a defective TCR signalingmolecule due to a Zap70 mutation are less likely to differentiateinto Tfh than other Th cell subsets. In this regard, increased ex-pression levels of costimulatory molecules in a B6 background

may facilitate the development of Tfh by enhancing TCR sig-naling and compensating for defective TCR signaling caused bythe Zap70 mutation.Alteration of TCR signaling by a Zap70 mutation may not only

affect Tfh but also the differentiation and function of Tregs.Agonistic TCR signaling is required for Treg differentiation inthe thymus and their suppressive function in the periphery (45).In SKG mice, the number of Tregs is increased irrespective ofgenetic background, although their suppressive function is im-paired due to defective TCR signaling (10). Interestingly, sub-populations of Tregs and Tfrs in B6SKG mice lost the expressionof the typical Treg marker CD25. We previously reported that asubpopulation of Tfr localized in the GC lost CD25 expression butretained similar suppressive function to CD25+ Tfr (32), whereasanother report suggested that CD252 Tregs tended to lose Foxp3expression and trans-differentiated into Tfh (46). It remains to bedetermined how these alterations in Treg phenotypes in B6SKGmice contribute to the development of lupus-like autoimmunedisease.In addition to SKG mice, other mutations of TCR-proximal

signaling such as mutations of Zap70 or the CD3z chain lead toa variety of phenotypes (11, 47, 48). Partial attenuation of TCRsignaling by Zap70 mutations leads to autoimmune disease de-velopment or autoantibody production, depending on the mutationtype and the genetic background of the mice (47, 48), whereasZap70 deficiency leads to a lack of mature CD4 T cells and im-munodeficiency. In CD3z knockout mice (Cd2472/2), TCR sig-naling is severely impaired but not completely inhibited becausethe signal can be transmitted via a CD3 complex other than thatof the z family (49). Interestingly, CD3z knockout mice developmultiorgan inflammation accompanied by Th1 infiltration but failto develop autoantibody production (50). Thus, there appears to bea distinct threshold for the attenuation of TCR signaling to de-velop a systemic autoimmune disease characterized by autoanti-body production. Although TCR signaling should be defectivewithin a certain range for self-reactive T cells to escape thymicnegative selection, a certain strength of TCR signaling is requiredfor their differentiation into Tfh in the periphery. Partiallyimpaired TCR signal transduction that meets these central andperipheral activation thresholds may provoke the final mani-festation of systemic autoimmune disease characterized byTfh and GC development.Recent genome-wide linkage analyses revealed that multiple

genes in the TCR signaling pathway are associated with a widevariety of autoimmune diseases, including lupus. Among them,a polymorphism in protein tyrosine phosphatase-22 (PTPN22)is known to be one of the strongest risk factors associated withvarious autoimmune diseases, including SLE, RA, type 1 diabetes,and autoimmune thyroiditis (51). PTPN22 encodes the TCR-associated protein tyrosine kinases and potently suppressesTCR signaling through the dephosphorylation of Lck, Fyn, orZAP70. However, it remains controversial as to whether thedisease-associated PTPN22 polymorphisms reduce or enhanceTCR signaling (51). Several reports suggest that the PTPN22W620 mutation is a gain-of-function mutation that reducesTCR-proximal signaling, whereas other reports suggest that this is

B cells after the inhibition of costimulatory molecules after 2-wk treatment with anti-OX40L, anti-ICOSL, and CTLA-4 Ig (up to 500 mg/body for 2 wk).

(F) Proportion of Tfh among CD4 T cells and proportion of GC B cells among B220+ AA4.12 mature B cells after 2-wk treatment with anti-OX40L, anti-ICOSL,

and CTLA-4 Ig (up to 500 mg/body, for 2 wk) (n = 5 each). (G) Immunohistochemistry of IgG and C3. Kidney sections of B6SKG mice with or without CTLA4-Ig

treatment were stained with IgG and C3. Scale bar, 20 mm (n = 4 each). (H) Area intensity of IgG and C3 fluorescence in the kidney of B6SKGmice. The integrated

brightness of IgG and C3 deposition was analyzed by an all-in-one fluorescence microscope (BZ-700; KEYENCE). Five glomeruli per mouse were analyzed. Flow

cytometric data (C, D, and E) are representative of at least three independent experiments. **p , 0.01 versus control mice, Student t test.

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a loss-of-function mutation that causes hyperresponsive TCR sig-naling (51–53). Defective TCR signaling in the thymus may helpthe survival of self-reactive T cells in the periphery (8), whereasenhanced TCR signaling in the periphery may help their differ-entiation into Tfh (53). Thus, the effect of the alteration in TCRsignaling for the development of systemic autoimmunity shouldbe studied in further detail, considering both its influence in thethymus and in the periphery.In conclusion, we established a novel animal model of SLE with

a defect in TCR-proximal signaling, which develops lupus-likesystemic autoimmune disease in a strain-specific manner. Themodel will be useful to study the pathogenesis of SLE and todetermine how alterations in TCR-proximal signaling contributeto the development of systemic autoimmune disease under theinfluence of genetic and environmental factors.

AcknowledgmentsWe thank Atsuko Tamamoto and Sumie Nakagawa for technical assistance.

DisclosuresThis study was supported in part by a research grant from Bristol-Myers

Squibb and ONO Pharma (to M. Hashimoto). M. Hashimoto receives

honoraria from Bristol-Meyers Squibb. The other authors have no financial

conflicts of interest.

References1. Tsokos, G. C. 2011. Systemic lupus erythematosus. N. Engl. J. Med. 365:

2110–2121.2. Kammer, G. M., A. Perl, B. C. Richardson, and G. C. Tsokos. 2002. Abnormal

T cell signal transduction in systemic lupus erythematosus. Arthritis Rheum. 46:1139–1154.

3. Liossis, S. N., X. Z. Ding, G. J. Dennis, and G. C. Tsokos. 1998. Altered patternof TCR/CD3-mediated protein-tyrosyl phosphorylation in T cells from patientswith systemic lupus erythematosus. Deficient expression of the T cell receptorzeta chain. J. Clin. Invest. 101: 1448–1457.

4. Takeuchi, T., K. Suzuki, T. Kondo, K. Yoshimoto, and K. Tsuzaka. 2012. CD3z defects in systemic lupus erythematosus. Ann. Rheum. Dis. 71(Suppl. 2):i78–i81.

5. Baniyash, M. 2004. TCR zeta-chain downregulation: curtailing an excessiveinflammatory immune response. Nat. Rev. Immunol. 4: 675–687.

6. Majeti, R., Z. Xu, T. G. Parslow, J. L. Olson, D. I. Daikh, N. Killeen, andA. Weiss. 2000. An inactivating point mutation in the inhibitory wedge of CD45causes lymphoproliferation and autoimmunity. Cell 103: 1059–1070.

7. Yu, C. C., T. S. Yen, C. A. Lowell, and A. L. DeFranco. 2001. Lupus-like kidneydisease in mice deficient in the Src family tyrosine kinases Lyn and Fyn. Curr.Biol. 11: 34–38.

8. Sakaguchi, N., T. Takahashi, H. Hata, T. Nomura, T. Tagami, S. Yamazaki,T. Sakihama, T. Matsutani, I. Negishi, S. Nakatsuru, and S. Sakaguchi. 2003.Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causesautoimmune arthritis in mice. Nature 426: 454–460.

9. Drappa, J., L. A. Kamen, E. Chan, M. Georgiev, D. Ashany, F. Marti, andP. D. King. 2003. Impaired T cell death and lupus-like autoimmunity in T cell-specific adapter protein-deficient mice. J. Exp. Med. 198: 809–821.

10. Tanaka, S., S. Maeda, M. Hashimoto, C. Fujimori, Y. Ito, S. Teradaira, K. Hirota,H. Yoshitomi, T. Katakai, A. Shimizu, et al. 2010. Graded attenuation of TCRsignaling elicits distinct autoimmune diseases by altering thymic T cell selectionand regulatory T cell function. J. Immunol. 185: 2295–2305.

11. Hirota, K., M. Hashimoto, H. Yoshitomi, S. Tanaka, T. Nomura, T. Yamaguchi,Y. Iwakura, N. Sakaguchi, and S. Sakaguchi. 2007. T cell self-reactivity forms acytokine milieu for spontaneous development of IL-17+ Th cells that causeautoimmune arthritis. J. Exp. Med. 204: 41–47.

12. Hashimoto, M., K. Hirota, H. Yoshitomi, S. Maeda, S. Teradaira, S. Akizuki,P. Prieto-Martin, T. Nomura, N. Sakaguchi, J. Kohl, et al. 2010. Complementdrives Th17 cell differentiation and triggers autoimmune arthritis. J. Exp. Med.207: 1135–1143.

13. Bolland, S., and J. V. Ravetch. 2000. Spontaneous autoimmune disease inFc(gamma)RIIB-deficient mice results from strain-specific epistasis. Immunity13: 277–285.

14. Nishimura, H., M. Nose, H. Hiai, N. Minato, and T. Honjo. 1999. Developmentof lupus-like autoimmune diseases by disruption of the PD-1 gene encoding anITIM motif-carrying immunoreceptor. Immunity 11: 141–151.

15. Nishimura, H., T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori,S. Sasayama, A. Mizoguchi, H. Hiai, N. Minato, and T. Honjo. 2001. Autoim-mune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 291:319–322.

16. Strasser, A., S. Whittingham, D. L. Vaux, M. L. Bath, J. M. Adams, S. Cory, andA. W. Harris. 1991. Enforced BCL2 expression in B-lymphoid cells prolongs

antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA88: 8661–8665.

17. Kuo, P., M. Bynoe, and B. Diamond. 1999. Crossreactive B cells are presentduring a primary but not secondary response in BALB/c mice expressing a bcl-2transgene. Mol. Immunol. 36: 471–479.

18. Fukuyama, H., F. Nimmerjahn, and J. V. Ravetch. 2005. The inhibitoryFcgamma receptor modulates autoimmunity by limiting the accumulation ofimmunoglobulin G+ anti-DNA plasma cells. Nat. Immunol. 6: 99–106.

19. Segal, R., B. L. Bermas, M. Dayan, F. Kalush, G. M. Shearer, and E. Mozes.1997. Kinetics of cytokine production in experimental systemic lupus eryth-ematosus: involvement of T helper cell 1/T helper cell 2-type cytokines in dis-ease. J. Immunol. 158: 3009–3016.

20. De Maeyer-Guignard, J., F. Dandoy, D. W. Bailey, and E. De Maeyer. 1986.Interferon structural genes do not participate in quantitative regulation of in-terferon production by if loci as shown in C57BL/6 mice that are congenic withBALB/c mice at the alpha interferon gene cluster. J. Virol. 58: 743–747.

21. Lauwerys, B. R., J. Ducreux, and F. A. Houssiau. 2014. Type I interferonblockade in systemic lupus erythematosus: where do we stand? Rheumatology(Oxford) 53: 1369–1376.

22. Liu, T., T. Matsuguchi, N. Tsuboi, T. Yajima, and Y. Yoshikai. 2002. Differencesin expression of toll-like receptors and their reactivities in dendritic cells inBALB/c and C57BL/6 mice. Infect. Immun. 70: 6638–6645.

23. Yoshitomi, H., N. Sakaguchi, K. Kobayashi, G. D. Brown, T. Tagami,T. Sakihama, K. Hirota, S. Tanaka, T. Nomura, I. Miki, et al. 2005. A role forfungal beta-glucans and their receptor Dectin-1 in the induction of autoimmunearthritis in genetically susceptible mice. J. Exp. Med. 201: 949–960.

24. Marshak-Rothstein, A. 2006. Toll-like receptors in systemic autoimmune dis-ease. Nat. Rev. Immunol. 6: 823–835.

25. Cavallo, T., and N. A. Granholm. 1990. Bacterial lipopolysaccharide transformsmesangial into proliferative lupus nephritis without interfering with processingof pathogenic immune complexes in NZB/W mice. Am. J. Pathol. 137: 971–978.

26. Patole, P. S., H. J. Grone, S. Segerer, R. Ciubar, E. Belemezova, A. Henger,M. Kretzler, D. Schlondorff, and H. J. Anders. 2005. Viral double-stranded RNAaggravates lupus nephritis through toll-like receptor 3 on glomerular mesangialcells and antigen-presenting cells. J. Am. Soc. Nephrol. 16: 1326–1338.

27. Pawar, R. D., P. S. Patole, D. Zecher, S. Segerer, M. Kretzler, D. Schlondorff,and H.-J. Anders. 2006. Toll-like receptor-7 modulates immune complex glo-merulonephritis. J. Am. Soc. Nephrol. 17: 141–149.

28. Pawar, R. D., P. S. Patole, A. Ellwart, M. Lech, S. Segerer, D. Schlondorff, andH.-J. Anders. 2006. Ligands to nucleic acid-specific toll-like receptors and theonset of lupus nephritis. J. Am. Soc. Nephrol. 17: 3365–3373.

29. Guerard, S., M. Boieri, M. Hultqvist, R. Holmdahl, and K. Wing. 2016. TheSKG mutation in ZAP-70 also confers arthritis susceptibility in C57 Blackmouse strains. Scand. J. Immunol. 84: 3–11.

30. Linterman, M. A., W. Pierson, S. K. Lee, A. Kallies, S. Kawamoto, T. F. Rayner,M. Srivastava, D. P. Divekar, L. Beaton, J. J. Hogan, et al. 2011. Foxp3+ follicularregulatory T cells control the germinal center response. Nat. Med. 17: 975–982.

31. Chung, Y., S. Tanaka, F. Chu, R. I. Nurieva, G. J. Martinez, S. Rawal,Y. H. Wang, H. Lim, J. M. Reynolds, X. H. Zhou, et al. 2011. Follicular regu-latory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions.Nat. Med. 17: 983–988.

32. Wing, J. B., Y. Kitagawa, M. Locci, H. Hume, C. Tay, T. Morita, Y. Kidani,K. Matsuda, T. Inoue, T. Kurosaki, et al. 2017. A distinct subpopulation ofCD25- T-follicular regulatory cells localizes in the germinal centers. Proc. Natl.Acad. Sci. USA 114: E6400–E6409.

33. Crotty, S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29:621–663.

34. Vinuesa, C. G., M. C. Cook, C. Angelucci, V. Athanasopoulos, L. Rui,K. M. Hill, D. Yu, H. Domaschenz, B. Whittle, T. Lambe, et al. 2005. A RING-type ubiquitin ligase family member required to repress follicular helper T cellsand autoimmunity. Nature 435: 452–458.

35. Ding, Y., J. Li, P. Yang, B. Luo, Q. Wu, A. J. Zajac, O. Wildner, H. C. Hsu, andJ. D. Mountz. 2014. Interleukin-21 promotes germinal center reaction byskewing the follicular regulatory T cell to follicular helper T cell balance inautoimmune BXD2 mice. Arthritis Rheumatol. 66: 2601–2612.

36. Choi, J. Y., J. H. Ho, S. G. Pasoto, V. Bunin, S. T. Kim, S. Carrasco, E. F. Borba,C. R. Goncalves, P. R. Costa, E. G. Kallas, et al. 2015. Circulating follicularhelper-like T cells in systemic lupus erythematosus: association with diseaseactivity. Arthritis Rheumatol. 67: 988–999.

37. Poholek, A. C., K. Hansen, S. G. Hernandez, D. Eto, A. Chandele, J. S. Weinstein,X. Dong, J. M. Odegard, S. M. Kaech, A. L. Dent, et al. 2010. In vivo regulation ofBcl6 and T follicular helper cell development. J. Immunol. 185: 313–326.

38. Ferguson, S. E., S. Han, G. Kelsoe, and C. B. Thompson. 1996. CD28 is requiredfor germinal center formation. J. Immunol. 156: 4576–4581.

39. Akiba, H., K. Takeda, Y. Kojima, Y. Usui, N. Harada, T. Yamazaki, J. Ma,K. Tezuka, H. Yagita, and K. Okumura. 2005. The role of ICOS in the CXCR5+follicular B helper T cell maintenance in vivo. J. Immunol. 175: 2340–2348.

40. Mihara, M., I. Tan, Y. Chuzhin, B. Reddy, L. Budhai, A. Holzer, Y. Gu, andA. Davidson. 2000. CTLA4Ig inhibits T cell-dependent B-cell maturation inmurine systemic lupus erythematosus. J. Clin. Invest. 106: 91–101.

41. Teichmann, L. L., J. L. Cullen, M. Kashgarian, C. Dong, J. Craft, andM. J. Shlomchik. 2015. Local triggering of the ICOS coreceptor by CD11c(+)myeloid cells drives organ inflammation in lupus. Immunity 42: 552–565.

42. Choi, Y. S., R. Kageyama, D. Eto, T. C. Escobar, R. J. Johnston, L. Monticelli,C. Lao, and S. Crotty. 2011. ICOS receptor instructs T follicular helper cellversus effector cell differentiation via induction of the transcriptional repressorBcl6. Immunity 34: 932–946.

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nloaded from

43. Jacquemin, C., N. Schmitt, C. Contin-Bordes, Y. Liu, P. Narayanan, J. Seneschal,T. Maurouard, D. Dougall, E. S. Davizon, H. Dumortier, et al. 2015. OX40 li-gand contributes to human lupus pathogenesis by promoting T follicularhelper response. Immunity 42: 1159–1170.

44. Fazilleau, N., L. J. McHeyzer-Williams, H. Rosen, and M. G. McHeyzer-Williams.2009. The function of follicular helper T cells is regulated by the strength ofT cell antigen receptor binding. Nat. Immunol. 10: 375–384.

45. Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cellsand immune tolerance. Cell 133: 775–787.

46. Tsuji, M., N. Komatsu, S. Kawamoto, K. Suzuki, O. Kanagawa, T. Honjo,S. Hori, and S. Fagarasan. 2009. Preferential generation of follicular B helperT cells from Foxp3+ T cells in gut Peyer’s patches. Science 323: 1488–1492.

47. Sakaguchi, S., S. Tanaka, A. Tanaka, Y. Ito, S. Maeda, N. Sakaguchi, andM. Hashimoto. 2011. Thymus, innate immunity and autoimmune arthritis:interplay of gene and environment. FEBS Lett. 585: 3633–3639.

48. Au-Yeung, B. B., N. H. Shah, L. Shen, and A. Weiss. 2018. ZAP-70 in signaling,biology, and disease. Annu. Rev. Immunol. 36: 127–156.

49. Shores, E. W., M. Ono, T. Kawabe, C. L. Sommers, T. Tran, K. Lui, M. C. Udey,J. Ravetch, and P. E. Love. 1998. T cell development in mice lacking all T cellreceptor zeta family members (Zeta, eta, and FcepsilonRIgamma). J. Exp. Med.187: 1093–1101.

50. Deng, G. M., J. Beltran, C. Chen, C. Terhorst, and G. C. Tsokos. 2013. T cellCD3z deficiency enables multiorgan tissue inflammation. J. Immunol. 191:3563–3567.

51. Burn, G. L., L. Svensson, C. Sanchez-Blanco, M. Saini, and A. P. Cope. 2011.Why is PTPN22 a good candidate susceptibility gene for autoimmune disease?FEBS Lett. 585: 3689–3698.

52. Vang, T., M. Congia, M. D. Macis, L. Musumeci, V. Orru, P. Zavattari,K. Nika, L. Tautz, K. Tasken, F. Cucca, et al. 2005. Autoimmune-associatedlymphoid tyrosine phosphatase is a gain-of-function variant. Nat. Genet. 37:1317–1319.

53. Maine, C. J., K. Marquardt, J. Cheung, and L. A. Sherman. 2014. PTPN22controls the germinal center by influencing the numbers and activity ofT follicular helper cells. J. Immunol. 192: 1415–1424.

3172 STRAIN-SPECIFIC LUPUS-LIKE SYSTEMIC AUTOIMMUNITY

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3 months old 12 months old 12 months old3 months old

Supplemental Figure 1. Histology of the joint, pancreas, and skin tissues of B6SKG mice.(A–C) Hematoxylin-eosin staining of tissue sections of B6WT and B6SKG mice at 3 months and 12 months of age.

(A) Ankle joint sections. The chondrocyte layers are rough, and an irregular bone layer exists in B6SKG mice but not in

B6WT mice at 3 months and 12 months of age. (B) Pancreas section. Inflammation in Langerhans islets is observed in

B6SKG mice at 12 months of age, but not in B6WT mice. (C) Skin section. No abnormal findings were detected in the

epidermis or dermis between B6SKG and B6WT mice at 3 months of age. Folliculitis was observed in the dermis of

B6SKG mice, but not B6WT mice at 12 months of age. Scale bars in A, 50 μm; scale bar in B and C, and 20 μm.

Imiquimod CpG Poly(I;C)

B6WT B6SKG B6WT B6SKG B6WT B6SKG

MannanArthritis score

* *

B

A

LPS Mannan Zymosan

B6WT B6SKG B6WT B6SKG B6WT B6SKG

mIU/ml

SKGBALB/c B6WT B6SKG

NS NS NS

NS

0

100

200

300

400

500

600

700

0

100

200

300

400

500

0

100

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0

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100

120

0

10

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60

0

1

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4

5

6

Supplemental Figure 2. Anti-dsDNA antibody production and joint scores after

innate immune stimulation.

(A) ELISA of anti-dsDNA antibody IgG after innate immune stimulation. B6WT mice and B6SKG mice

with an anti-dsDNA antibody titer of <100 U/ml before treatment were selected. For the TLR stimulation

experiment, the mice were subcutaneously implanted with an osmotic pump for 14–28 days containing

various TLR agonists: 700 μg Poly(I;C), 280 μg LPS, 280 μg imiquimod, and 280 μg CpG-ODN1668.

Zymosan A and mannan were injected intraperitoneally twice a week up to 2 mg and 20 mg, respectively,

for 28 days; n = 5 each. *P < 0.05, **P < 0.01 vs. control mice, Student’ s t-test. (B) Joint scores after

mannan treatment. B6SKG mice developed slight arthritis compared with SKG mice; n = 5 each.

%

SP CD4 SP CD8

DN3 DN4

** ** **

A B DNs

DPs

** **

CD4

CD8

C

24.4

11.3

9.5

2.7

SKGBALB/c

CD44

CD62L

DSKGBALB/c

E

M

N

6.9

69.3

15.8

7.847.7

BALB/c

SKG

Balb/c SKG0

1020304050607080

Balb/c SKG0

10

20

30

40

50

Balb/c SKG

808284868890929496

Balb/c SKG

02468

101214

Balb/c SKG

0

1

2

3

4

5

Balb/c SKG

*

0

0.5

1

1.5

2

2.5

4.6

Supplemental Figure 3. Thymic selection in SKG mice.(A) Hematoxylin–eosin staining of the thymic gland in 28–35-day-old BALB/c and SKG mice. (B) Flow cytometric analysis of the maturation of

developing T cells in the thymus; n = 5 each. *P < 0.05, **P < 0.01 vs. control mice, Student’ s t-test. (C) Flow cytometric analysis of CD4 and

CD8 on lymphocytes in the spleen. (D) Flow cytometric analysis of CD44 and CD62L gated on CD3+ CD4+ T cells in the spleen.

%

IL-6/HPRT

**

A BCD11c DC

0

1

2

3

4

5

6

7

**RQ

3

2

1

0SKGBalb/c B6WT B6SKG

**NS NS

NS

SKGBalb/c B6WT B6SKG

2.9

3.9

1.8

1.2

36.7

33.0

13.4

10.4

44.6

36.8

30.4

23.0%

0

10

20

30

40

50

CD80

SKGBALB/c B6WT B6SKG0

10

20

30

40

50

60

CD86

SKGBALB/c B6WT B6SKG

39.7

22.6

44.5

16.5

DC

0

10

20

30

40

50

SKGBALB/c B6WT B6SKG

ICOSL

0

1

2

3

4

5

6

7

SKGBALB/c B6WT B6SKG

OX40L

E

1.5

1.5

2.2

2.1

90.2

87.6

88.2

85.3

4.3

4.4

5.0

4.8

19.4

26.7

22.3

28.4

%

CD80

SKGBALB/c B6WT B6SKG0

1

2

3

4

5

6

7

CD86

SKGBALB/c B6WT B6SKG0

5

10

15

20

25

30

F

ICOSL

SKGBALB/c B6WT B6SKG84

86

88

90

92

94

96

OX40L

SKGBALB/c B6WT B6SKG0

0.5

1

1.5

2

2.5

*

BALB/c

SKG

B6WT

B6SKG

BALB/c

SKG

B6WT

B6SKG

CD80 ICOSLCD86 OX40L

CD80 ICOSLCD86 OX40L

****

NS ****

NS

****

NS NSNS

NS

NSNS

NSNSNS

NS

NSNS

NSNS

NS

Supplemental Figure 4. Expression of costimulatory molecules of APCs (CD11c DC and B220 B cells).(A) Proportion of CD11chigh DCs in the spleen; n = 5 each. The proportion was significantly higher in B6WT mice than in BALB/c mice with the number further increased by the Zap70 mutation.

(B) Il-6 mRNA expression of splenic CD11c DCs; n = 3 each. No significant difference was observed among BALB/c, SKG, B6WT, or B6SKG mice. (C, D) Representative histograms (C) and %

positive cells (n = 5) (D) for CD80, CD86, ICOSL, and OX40L expression in CD11c DCs. The expression levels of CD80/86, IOCSL, but not OX40L were significantly higher in the DCs of B6WT

mice than in those of BALB/c mice. The levels of CD80/86 and ICOSL were upregulated on SKG mice compared with BALB/c mice, while no difference was found between B6SKG and B6WT

mice. (E, F) Representative histograms (E) and % positive cells (n=3) (F) for CD80, CD86, ICOSL, and OX40L expression on B cells. The expression levels were not different between BALB/c

and B6WT mice. ICOSL expression was down-regulated in B6SKG mice compared with B6WT mice.